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目的克隆乙型肝炎病毒(HBV)前-S1蛋白反式激活新基因PS1TP5的cDNA,并应用生物信息学技术初步探讨其结构及功能。方法应用聚合酶链反应(PCR)技术以HepG2细胞的cDNA为模板扩增PS1TP5,以pGEM-T载体进行TA克隆,通过PCR、限制性酶切分析及测序进行鉴定,再将其亚克隆到真核表达载体pcDNATM3.1/myc-HisA,通过PCR、限制性酶切分析进行鉴定,并应用生物信息学技术初步分析其物理化学性质、蛋白质结构和功能。结果PCR成功扩增出PS1TP5基因,并将其分别克隆进pGEM-T和pcDNATM3.1/myc-HisA载体,经PCR、限制性酶切鉴定后测序证实。因其可以被前-S1蛋白反式激活,故命名为前-S1反式激活蛋白5(PS1TP5),已在GenBank中注册,注册号AY427953。生物信息学分析确定其ORF为438个核苷酸(nt),编码产物为145个氨基酸残基(aa)。结论发现了HBV前-S1蛋白反式激活新基因PS1TP5,构建了pcDNATM3.1/myc-HisA真核表达载体,为进一步研究其生物学功能及慢性乙型肝炎发病机制创造了条件。
Objective To clone the cDNA of PS1TP5 transactivated by pre-S1 protein of hepatitis B virus (HBV) and to investigate its structure and function by bioinformatics. Methods Polymerase chain reaction (PCR) technique was used to amplify PS1TP5 from cDNA of HepG2 cells. TA cloning was carried out on pGEM-T vector and identified by PCR, restriction analysis and sequencing, and then subcloned into true The nuclear expression vector pcDNATM3.1 / myc-HisA was identified by PCR and restriction analysis, and its physicochemical properties, protein structure and function were analyzed by bioinformatics. Results The PS1TP5 gene was successfully amplified by PCR and cloned into pGEM-T and pcDNATM3.1 / myc-HisA vector respectively. The PCR products were confirmed by restriction enzyme digestion and sequencing. Because it can be trans-activated by pre-S1 protein, it is named pre-S1 transactivator 5 (PS1TP5) and has been registered in GenBank with accession number AY427953. Bioinformatics analysis confirmed that the ORF was 438 nucleotides (nt) and the encoded product was 145 amino acid residues (aa). CONCLUSION: The transactivation of pre-HBV protein PS1TP5 has been found and the eukaryotic expression vector pcDNATM3.1 / myc-HisA has been constructed, which provides conditions for further study on its biological function and pathogenesis of chronic hepatitis B.